The invention relates to a fermentative method of producing isobutanol from sugars.
Isobutanol has excellent properties as fuel. In addition, it is also a useful chemical e.g. as base chemical for the production of other chemicals or as solvent. Today, isobutanol is predominantly produced by petrochemical methods from fossil resources. However, a much more promising prospect would be to produce it from renewable resources such as e.g. vegetable sugars or vegetable waste. Recently, two microbial, non-fermentative methods were presented with which isobutanol can be produced from sugars (Atsumi et al., 2008; US patent application 2007/0092957). In both methods host cells were induced, by the insertion of heterologous DNA, to produce isobutanol and also other branch-chained alcohols from the metabolic intermediate pyruvate, which forms as a result of the breakdown of sugars. However, common to both described methods is that they are non-fermentative, i.e. their redox balances are not equilibrated when sugars break down into isobutanol. They can therefore only be used in complex media by simultaneous conversion of co-substrates, through the formation of by-products or under aerobic conditions. This greatly reduces the practicability of the methods and makes them economically unappealing.
One solution would be the development of a fermentative microbial process which could take place in minimal media, without co-substrates and also under anaerobic or oxygen-limited conditions. In particular yeasts and in particular those of the genus Saccharomyces such as e.g. Saccharomyces cerevisiae would be suitable as microorganisms. Interestingly, yeasts already have all the enzymes that are necessary for the formation of isobutanol from sugars. However, these enzymes are located in different compartments of the yeast cells (cytosol and mitochondria), they use different co-factors that are not or not effectively convertible into one another (NAD+/NADH and NADP+/NADPH) and the enzymes are expressed only weakly or under special conditions or have a low enzyme activity. In order to achieve an effective production of isobutanol from sugars, the metabolic pathways present would have to be modified such that with their help isobutanol could be produced in redox-neutral manner and with energy gain in the form of ATP, including under anaerobic or oxygen-limited conditions. The development of such a fermentative method of producing isobutanol from sugars is the object and aim of this invention.
Sugars such as e.g. glucose are broken down into pyruvate in host cells such as e.g. yeasts predominantly through the metabolic pathway of glycolysis. Two molecules of pyruvate are produced from one molecule of glucose. In addition, 2 energy-rich compounds are produced in the form of ATP and 2 molecules of NAD+ are reduced to NADH+H+. Pyruvate is then usually converted to ethanol either by the pyruvate decarboxylases and alcohol dehydrogenases or it is transported into the mitochondria, where it is converted into acetyl-CoA by pyruvate dehydrogenase and finally funneled into the citric acid cycle. In addition, pyruvate can also be converted in some other reactions. One of these reaction paths is the biosynthetic pathway to the amino acid valine. On the other hand, however, valine can also be broken down i.a. into the product isobutanol. If the biosynthetic pathway and the catabolic path of valine could be shortened, isobutanol could then be produced direct from sugars via pyruvate. Such a metabolic pathway combines the enzymes which are involved in the biosynthesis of valine (from pyruvate to α-ketoisovalerate) with those which are involved in valine breakdown (from α-ketoisovalerate to isobutanol). The yeast Saccharomyces cerevisiae itself contains all the genes required for this. ILV2 (YMR108W) (SEQ. ID. no. 1) encodes the acetolactate synthase which converts two pyruvate molecules into acetolactate. The Ilv2 enzyme (SEQ. ID. no. 2) is activated by the Ilv6 protein (=YCL009C) (SEQ. ID. no. 4). ILV5 (YLR355C) (SEQ. ID. no. 5) encodes the acetohydroxy acid reducto-isomerase which converts acetolactate into 2,3-dihydroxy isovalerate. ILV3 (YJR016C) (SEQ. ID. no. 7) encodes the dihydroxy acid dehydratase which converts 2,3-dihydroxy isovalerate into 2-ketoisovalerate. 2-ketoisovalerate is then usually transaminated into valine; by the transaminases Bat1 (SEQ. ID. no. 10) and Bat2 (SEQ. ID. no. 12). But if this reaction is bypassed or reduced, 2-ketoisovalerate could then also be converted by different 2-keto acid decarboxylases into isobutyraldehyde, e.g. by the enzymes Pdc1 (SEQ. ID. no. 14), Pdc5 (SEQ. ID. no. 16), Pdc6 (SEQ. ID. no. 18), Aro10 (SEQ. ID. no. 20), Thi3 (SEQ. ID. no. 22) (Dickinson et al., 1998; 2003). This direct conversion is usually impeded inter alia by the different compartmentalization of the enzymes (mitochondria, cytosol). Isobutyraldehyde can then finally be reduced to isobutanol by different alcohol dehydrogenases (Dickinson et al., 2003). These include i.a. Adh1-7 (SEQ. ID. no. 24), (SEQ. ID. no. 26), (SEQ. ID. no. 28), (SEQ. ID. no. 30), (SEQ. ID. no. 32), (SEQ. ID. no. 34), (SEQ. ID. no. 36), Sfa1 (SEQ. ID. no. 38), Ypr1 (SEQ. ID. no. 40).
However, most of the named enzymes are not strongly enough expressed or have low levels of enzyme activity for an efficient production of isobutanol from pyruvate or sugars. Another problem is the co-factor specificity and redox balance. During the reduction of the two molecules of pyruvate produced from glycolysis to isobutanol, one molecule of NADPH from the acetohydroxy acid reducto-isomerase and one molecule of NADH or NADPH from the branch-chained alcohol dehydrogenases are required. However, in glycolysis, two molecules of NADH are produced from one molecule of glucose in the glyceraldehyde-3-phosphate dehydrogenase reaction. Thus there is a shortfall of NADPH and an excess of NADH. But NADH is not easily convertible into NADPH. On the other hand, the enzymes Ilv2/Ilv6, Ilv5 (SEQ. ID. no. 6) and Ilv3 (SEQ. ID. no. 8) are at least mainly located in the mitochondria of the yeast cells. The pyruvate must therefore firstly be transported into the mitochondria and finally the 2-ketoisovalerate transported out of the mitochondria into the cytosol. As transport via membranes can often have a limiting effect on flows of material, it would therefore be desirable to shift all reactions into the cytosol. Equally disadvantageous for an efficient production of isobutanol is that some intermediates are drawn off for other metabolic reactions on the way from the sugar to the product. This applies above all to pyruvate which is largely converted to ethanol by the pyruvate decarboxylases and alcohol dehydrogenases. It is therefore important for a more efficient production of isobutanol to reduce or completely eliminate these secondary reactions.
The object and aim of this invention is therefore to provide a fermentative method of producing isobutanol from sugars in which (i) the yeast's own set of enzymes is used for the metabolic pathway from pyruvate to isobutanol by increasing their expression or activities, i.e. without heterologous genes having to be introduced into the yeast, (ii)a) the co-factor specificity of acetohydroxy acid reducto-isomerase is modified such that this enzyme preferably uses NADH instead of NADPH as a co-factor, or (ii)b) the co-factor specificity of the glyceraldehyde-3-phosphate dehydrogenase is modified such that this enzyme preferably uses NADP+ instead of NAD+ as co-factor or a heterologous NADP glyceraldehyde-3-phosphate dehydrogenase is expressed in the yeast cells, (iii) the formation of secondary products such as e.g. ethanol is minimized and (iv) in which as many of the enzymes involved as possible are located in the cytosol of the yeast cells.
The object is achieved according to the invention by the over-expression of the enzyme activities of Ilv2 with or without its activator Ilv6, Ilv5, Ilv3, at least one 2-keto acid decarboxylase such as e.g. Aro10 and at least one alcohol dehydrogenase which can also reduce isobutyraldehyde (preferably Adh1 or Adh6, but also Adh2-5, Sfa1, Ypr1 or others). This is carried out firstly through the exchange of the respective promoters of the corresponding genes for stronger promoters, preferably, but not exclusively, constitutive promoters. Preferably, but not exclusively, promoter sequences are selected from HXT7, shortened HXT7, PFK1, FBA1, TPI1, PGK1, PMA1, ADH1, TDH3. Furthermore, the corresponding nucleic acid sequences of the genes are converted into codon-optimized alleles. Every amino acid is encoded at gene level by a codon. However, for most amino acids there are several different codons which code for a single amino acid. The genetic code is consequently degenerated. The preferred codon choice for a corresponding amino acid differs from organism to organism. Thus in the case of heterologously expressed genes, problems can occur if the host organism or the host cell has a very different codon usage. The gene may not be expressed at all, or only slowly. But a different codon usage can also be detected in genes of different proteins and metabolic pathways within a cell. Glycolysis genes from S. cerevisiae are known to be strongly expressed. They have a strongly restrictive codon usage which corresponds approximately to the quantity ratios of the corresponding tRNAs. The adaptation of the codon usage of the genes ILV2, (ILV6) (SEQ. ID. no. 3), ILV5, ILV3, one of the above-named 2-keto acid decarboxylase genes and one of the above-named alcohol dehydrogenase genes to the preferred codon usage of S. cerevisiae results in an improvement of the isobutanol formation rate in yeast. The preferred codon usage can be defined as described in Wiedemann and Boles (2008) for the glycolytic genes, but need not necessarily be restricted to these examples. The over-expressed, possibly codon-optimized genes can either be inserted cloned on plasmids into the yeast cells, they can be integrated into the genome of the yeast cells or they can genomically replace the naturally occurring alleles.